1. Field of the Invention
A fiber-optic biosensor detects pathogens based on immunoassay. Acoustic Enhancement concentrates the bound antibody enhancing detection.
2. Description of the Relevant Art
Sensitive detection of fluorescence or luminescence from biological reaction in vivo is important for many biosensor applications. Considerable effect has been devoted to the development of fiber-optic fluorescent biosensors because of their potential sensitivity, detection speed, and applicability to a wide variety of assay conditions.
Fiber-optic fluorescent immunosensors require that the dye molecules, which indicate unambiguously the presence of the antigen, find their way to the optically active region of the fiber. This can be accomplished by immobilizing the capture antibodies directly on the tip or the tapered core of the fiber, but this means that the capture process is localized and that several rinsing steps are needed to avoid ambiguity and to recycle the system for another measurement. On the other hand, performing the immunoassay on microparticles that are distributed throughout the sample cell and that are subsequently concentrated into the fiber""s sensing volume promises greatly improved efficiency. Two well-studied techniques for manipulating microparticles that could be used for this purpose are based on magnetism and n acoustics. The present invention is based in part of the latter technique.
It has long been known that small particles and biological cells suspended in a liquid can be trapped in the force field generated by a stationary ultrasonic wave. The acoustic radiation force on a rigid sphere or on a compliant sphere has been studied by many authors, Chen, X. and Apfel, R. E., Radiation force on a spherical object in an axisymmetric wave field and its application to the calibration of high-frequency transducers, J. Acoust. Soc. Am, 99, 1996, 713-724; and Wu, J. and Du, G., Acoustic radiation force on a small compressible sphere in a focused beam, J. Acoust. Soc. Am. 87, 1990, 997-1003. It is known that the average force that acts on a small compressive particle as a function of the mean square fluctuation of the pressure and velocity at the point where the particle is located. The results have been applied in many biological applications, such as rapid agglutination testing; Grundy, M. A., Moore, K. and Coakley, W. T., Increased sensitivity of diagnostic latex agglutination tests in an ultrasonic standing wave field, J. Immunological Methods, 176, 1994, 169-177; determination of the properties of red blood cells; Weiser, M. A. H. and Apfel, R. E., Extension of acoustic levitation to include the study of micron-size particles in a more compressible host liquid, J. Acoust. Soc. Am., 71, 1982, 1261-1268; and the concentration and separation of particles and cells; Whitworth, Glenn and Coakley, W. T., Particle column formation in a stationary ultrasonic field, J. Acoust. Soc. Am., 91, 1992, 79-85; and Coakley, W. T., Whitworth, G., Grundy, M. A., Gould, R. K. and Allman, R., Ultrasonic manipulation of particles and cells, Bioseparation, 4, 1994, 73-83; and Yasua, K., Kiyama, M. and Umemura, S. Deoxyribonucleic acid concentration using acoustic radiation force, J. Acoust. Soc. Am., 99, 1992, 1248-1258. Ultrasonic radiation forces have provided a real-time, non-contact method to manipulate particles and cells.
In a plane standing wave, the particles that are stiffer and/or denser than the surrounding medium gather in the nodal planes spaced one-half wavelength apart. For a standing plane wave confined to a cylindrical container with a rigid wall, if the cross-sectional profile of the sound field has a Besell or Gaussian shape intense on the axis, weak at the wall, then the potential energy of a suspended particle has a gradient in the radial direction. For particles denser than the fluid (xcfx81o greater than xcfx81), the force pushes them toward the axis, forming a striated column. For the formation of both layers and columns of particles, the forces are proportional to the acoustic intensity, suggesting improved results with increased sound amplitude and frequency. The intensity is limited, however, by the generation of cavitation and streaming, which destroy the formations.
Broadly the invention embodies acoustic complex manipulation for enhancement of the sensitivity of a fiber-optic biosensor using fluorescent immunoassay techniques for the rapid detection of a pathogen, e.g. Salmonella. In a preferred embodiment, a fiber-optic probe defines an axis in an acoustic chamber whereby the target complexes are first be formed in parallel layers perpendicular to the fiber and then are concentrated along the fiber for maximum sensitivity.
According to the teachings of the invention various immunoassays can be performed. One embodiment of the invention is a xe2x80x9csandwichxe2x80x9d assay in which the Salmonella cells are captured by antibodies that are attached to polystyrene microspheres. The Salmonella is made visible when it captures, in turn, free antibodies that have been labeled with a fluorescent dye. The entire microsphere-antibody-Salmonella-antibody complex is manipulated acoustically.
In another embodiment of the invention, simple Salmonella-antibody complexes are directly concentrated acoustically along the cell axis.